Chapter 5 94 Representative fluorescence images of the proximal tubule epithelial cells (circled structure) in a dynamin-knockdown zebrafish embryo. (C and D) the number of fluorescent droplets below the luminal surface were counted in control embryos, dynamin-knockdown embryos, and wildtype embryos injected with puromycin aminonucleoside (PAN) as a positive control for increased glomerular permeability. *p < 0.05 versus control. A digital high-pass filter has been placed over figures A and B to enhance contrast between reabsorption droplets and the surrounding tissue. The images in A and B were taken at the same magnification; scale bar = 10 µm. Dynamin protein levels are increased in the glomeruli of patients with proteinuria Finally, we examined the expression of dynamin in the kidneys of patients with proteinuria (Figures 3A through D). Patient characteristics are given in Table 3. Biopsies were obtained from patients with proteinuric kidney disease and control subjects, and stained by immunohistochemistry for the presence of dynamin. Positive staining was found primarily in podocytes, at the tubular epithelial brush border, and in endothelial cells of larger vessels. When comparing individual disease groups to the controls, the glomeruli of patients with minimal change disease (MCD) and lupus nephritis (LN) had a significantly higher dynamin-positive area percentage. (p = 0.03 and p = 0.04, respectively, Figure 3I). No scarred glomeruli were included in these analyses. No correlation was found between proteinuria levels and dynamin expression levels. Cathepsin L protein levels are also increased in the glomeruli of patients with proteinuria The same cohort was also stained by immunohistochemistry for the presence of cathepsin L (Figures 3E through H). We found a significantly higher glomerular cathepsin L-positive area percentage in patients with minimal change disease (MCD), lupus nephritis (LN), and IgA nephropathy (IgAN, p < 0.0001 compared to controls in all three groups, Figure 3J). A positive correlation was found between the glomerular positive area percentages for dynamin and cathepsin L (r = 0.928, p = 0.0075). Table 3. Patient characteristics Control DN LN MCD FSGS IgAN Number of patients (n) 15 6 6 8 5 4 Mean age, years (SD) 54,8 (16,11) 50,2 (19,11) 29,3 (12,15) 31,1 (20,88) 36,2 (7,19) 34,5 Mean serum creatinin, µmol/l (SD) 101,57 (37,95) 140,80 (76,30) 112,80 (39,51) 77,38 (27,77) 112,80 (60,24) 107,5 (34,09) Proteinuria, g/24 h (SD) 0,14 (0,12) 1,15 (0,82) 2,50 (1,86) 3,56 (3,32) 5,08 (1,84) 4,40 (3,19) Figure 2. Continued
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